Tobacco-related deaths cost the United States approximately $200 billion each year. Nicotine is singularly responsible for the dependence-forming properties of tobacco smoking and, once introduced to the body, rapidly affects the brain within 15 seconds. Breaking nicotine addiction is challenging and relapse rates remain high. The extremely rapid timescale of nicotine action renders existing techniques for studying nicotine accumulation in the brain (e.g., microdialysis, PET, radioimmunoassay) ill-suited for long-term addiction studies in freely moving animals. Biosensors are a proven technology for monitoring real-time changes in CNS neurochemical concentrations. The most critical component of a biosensor is the enzyme used as the biorecognition element, and no aspect of a biosensor's final design is as vital as a properly folded enzyme with sufficient activity and stability profiles. To date, no nicotine-specific enzyme has been reported. A primary goal of this Phase I SBIR proposal is to begin the transformation of an existing oxidase enzyme's kcat, stability at 37oC, Km, and Tm into an enzyme capable of the specific detection of nicotine - a process that will be completed during Phase II. We will combine structure-guided design with directed protein evolution (including a suitable selection process) to hone and optimize a new nicotine oxidase enzyme suitable as the foundation of a nicotine biosensor. The resulting nicotine biosensor promises to impact nicotine addiction studies by allowing real-time recordings of CNS nicotine concentrations in freely moving animals for up to one week. Furthermore, the nicotine biosensor could be used in conjunction with other biosensors, which will allow for the simultaneous monitoring of changes in nicotine concentration in tandem with other important CNS analytes (i.e., glucose, glutamate, lactate) or addictive compounds (i.e., ethanol). This approach promises to reveal new insights into nicotine distribution, dynamics and flux. Furthermore, a nicotine biosensor should find use as a screening tool for the development of new pharmacologic agents designed to assist in smoking cessation and inhibit relapse. The completion of both Phase I and Phase II components will provide two important innovations to the scientific community: 1) A nicotine biosensor suitable for long-term (i.e., up to one week) addiction studies that provides second-by-second changes of nicotine concentration in the CNS. 2) A refined approach for the development of new biosensors that target analytes important for addiction and for which no oxidase enzyme currently exists (i.e., cocaine and caffeine). PUBLIC HEALTH RELEVANCE: It is estimated that 50% of regular smokers die due to smoking-related complications. In addition, nicotine addiction associated with cigarette smoking takes a tremendous economic toll, costing the United States nearly $200 billion annually, including $97 billion in lost productivity and $96 billion in health care expenditures. This projec allows for a better understanding of nicotine addiction, which could advance smoking cessation research and potentially prevent 450,000 smoking-related deaths per year in the United States.